The invention concerns a process and a device for the detection of microscopically small objects, particularly for the detection of the presence and/or for the measurement of the location and/or the change in location of the objects, for example in fluidic microsystems. The microscopically small objects are particularly synthetic or biological particles which are manipulated in a fluidic microsystem. The invention also concerns uses of the process and/or the device, particularly for object detection depending on mechanical, electrical, or chemical interactions of the objects with their environment or other objects.
Using fluidic Microsystems for particle-specific manipulation of microscopically small objects under the effect of hydrodynamic and/or electrical forces is generally known. The manipulation of biological particles in microsystems with high frequency electric fields on the basis of negative dielectrophoresis is, for example, described by G. Fuhr et al. in xe2x80x9cNaturwissenschaftenxe2x80x9d, vol. 81, 1994, p. 528 et seq. The manipulation of the objects comprises, among other things, sorting according to specific characteristics, alteration of the object under the effect of electrical fields (e.g. cell poration), chemical treatment, mutual bringing together and interaction of the objects and similar actions. The provision of predetermined hydrodynamic and/or electrical forces is performed through the design of the channel structure of the microsystem and/or the geometric shapes of microelectrodes for forming high frequency electric fields and their control.
The previously known Microsystems do allow monitoring of the object manipulation occurring with optical means, e.g. using a microscope with a camera. This optical monitoring has, however, been restricted until now to visual examinations or the use of costly image recognition processes for processing the camera image. The image processing processes are, however, significantly too slow for the object speeds which arise, if real-time monitoring is to be practiced. Therefore, until now the implementation of automatic system controls, in which, for example, specific process variations occur depending on location, movement state, or number of objects observed, has been excluded.
A process for movement detection of microscopic objects which perform an at least partially periodic movement is known from PCT/EP97/07218. This process is based on the use of a Fourier analysis of a detector signal which characterizes the object movement over multiple movement periods. This process represents a significant simplification relative to the use of computer-aided image processing processes, but it is restricted to applications with periodic movements. In general, however, non-periodic movements or rest conditions of the objects also occur in a microsystem, the detection of which, particularly for automatic control of a microsystem, is also of interest.
Detecting the presence of small particles in suspensions on the basis of scattered light measurements is also known. However, this principle can only be realized with multiple particles, and can therefore not be particle-specific. In addition, no information on the movement state of particles (location, speed, or similar parameters) can be derived. It is the object of the invention to indicate an improved process for object detection which can be used in any desired movement or rest state of the objects to be detected and which allows rapid and reliable signal evaluation. The object of the invention is also to indicate devices for implementing such a process and new applications of the object detection according to the invention.
The invention is based on the idea of imaging the locational area (movement path or position) of the object to be detected enlarged on a mask which is set up in at least one segment for transmitting (reflection or transmission) of the light from one part of the locational area (so-called section) and otherwise for suppressing of transmission outside the segment. The at least one light-transmitting segment of the mask has a characteristic dimension which is smaller than the image of the entire object or the lateral extension of the movement path of the object. The quantities of light of the object image transmitted by the mask are summarily imaged on a detector device, at which a detector signal is generated having a predetermined relationship with the quantity of light detected and allowing an evaluation in regard to the presence of the object, its position, and/or the temporal change of the position. The quantity of light detected by the detector device is modulated in a characteristic way by the masked image of a section of the object and/or of the object path. The time dependence of the detector signal, particularly the temporal position of signal maxima, the amplitudes of the signal maxima, and the temporal amplitude shape in the vicinity of the signal maxima provides information not only on the location and the speed of the objects, but also on derived dimensions such as the frequency of periodic movements, quantitative information on particle numbers, movement direction, centering in the Microsystems, or similar dimensions.
One subject of the invention is therefore particularly object detection on the basis of a diaphragma measurement principle, in which a summary detection of the quantities of light coming out of a section of the object or the object path occurs. Correspondingly, the partial object imaging can, in principle, occur on a diaphragma (screen) with suitable dimension. In the simplest case, the mask consists of an element opaque to light with a round or angular opening which is suitably dimensioned for realization of the partial image of the object to be detected according to the invention.
However, corresponding to preferred embodiments of the invention, the mask has a segmentation with a predetermined geometry. The at least one segment for transmission of the light of the partial image has a geometric shape which is selected, depending on the application, according to the respective object movements (e.g. translation, rotation, translational vibration, rotational vibration, or similar movements) expected at the measurement point.
According to the invention, an enlarged image of the locational area of the objects to be detected (e.g. in a microsystem) is preferably provided on the mask. For this purpose, the segmentation of the mask, which is set up for transmission of the light from a partial image, can be absolutely larger than the section or part of the object concerned. This is advantageous for the production and justification of the mask.
Preferred applications of object detection according to the invention are provided in fluidic microsystems, particularly in automatic regulation of system functions, in dielectric single particle spectroscopy, and in the examination of interactions between objects and other objects and/or substrates. Objects detected according to the invention have a characteristic diameter in the range below 500 xcexcm down to the 100 nm range and comprise synthetic or biological particles (or particle aggregates). The synthetic particles are, for example, membrane-enclosed formations, such as liposomes or vesicles, or plastic particles (so-called beads). The biological particles are biological cells or cell aggregates or cell components, microorganisms, viruses, or similar objects.
One subject of the invention is also a device for implementation of object detection according to the invention, comprising an optical imaging unit for imaging a part of an object to be detected (or of its path) via a mask on a detector unit, which generates a detector signal in a specific relationship with the quantity of light detected, and an evaluation unit for determining characteristics of the movement or rest state of the object. The imaging unit contains, in particular, the mask, on which the object or its movement path is imaged in enlarged form and which only transmits a section of the image to the detector unit. According to a preferred embodiment of the invention, which is explained in detail below, the mask is a screen with a predetermined transmission geometry. The invention is, however, not restricted to this design, but can also be implemented with other mask designs which are set up to fulfill the same function as the transmitting screen. Instead of a transparent segment in an opaque mask material, an opaque segment (with the same geometry as the previously mentioned transparent segment) can be used as the mask in an otherwise transparent environment. Correspondingly, a reflection principle can be realized instead of the transmission principle.
The imaging unit is preferably part of a microscope arrangement, which is known per se, containing the mask for generating the partial image in the beam path. In this way, visual object observation can occur simultaneously with object detection. The combination with the microscope arrangement is, however, not urgently necessary. Particularly for automatic applications, the imaging unit can be provided directly in a microsystem.
The invention has the following advantages. The object detection according to the invention does not require imaging processing processes which are costly in measurement and time. It allows highly precise measurement with imaging and measurement units which are available per se. An existing microscope assembly can be set up for object detection according to the invention simply by attaching the mask mentioned. The mask segmentation according to the invention allows object detection without a large amount of adjustment. The object detection can be automated easily. Particular advantages result in the combination of mask-based detection with dielectric single particle spectroscopy for highly precise determination of dielectric particle characteristics from the movement characteristics of the particles in high frequency electric fields. Through the use of predetermined mask types, a device according to the invention can be adjusted without problems for the detection of greatly varying types of movement, without having to resort to image processing methods or having to perform a modification of the system. It is sufficient for a detector unit to contain one single detector whose measurement signals can be serially processed. This allows processing in real time, which is particularly significant for switching and sorting applications in microsystems.
A further advantage of the invention arises from the geometric mask structuring. In contrast to the typical xe2x80x9cpinholexe2x80x9d measurement principle, the mask segments are designed as smaller than the image of the entire object, but, unlike apertures (as they are known in, for example, confocal microscopy), are implemented as planar. In this way, increased functional reliability is achieved, even with deviations of the objects from the expected movement paths which occur in practice. The mask segmentation allows for compensation of tolerances as the object moves.